Hybrid immunoglobulin containing non-peptidyl linkage

Hybrid immunoglobulin containing non-peptidyl linkage
US11066459

The present invention provides a compound having the structure:
A-b - - - Z
wherein A is a biologically active structure of the compound; wherein Z is a protein component of the compound, which protein component comprises one or more polypeptides, wherein at least one of the one or more polypeptides comprises consecutive amino acids which (i) are identical to a stretch of consecutive amino acids present in a chain of an f_cdomain of an antibody; (ii) bind to an f_creceptor; and (iii) have at their n-terminus a sequence selected from the group consisting of a cysteine or selenocysteine; wherein the dashed line between b and Z represents a peptidyl linkage; and wherein the solid line between A and b represents a nonpeptidyl linkage, as well as intermediates dimers thereof, and processes of producing the compounds of the invention.

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Patent 11066459
Priority Mar 14 2014
Filed Mar 13 2015
Issued Jul 20 2021
Expiry Mar 13 2035
Inventors Capon, Dan…
Assg.orig BIOMOLECUL…
Assg.curr BIOMOLECUL…
Entity Small
Referenced by 0
References 29
Maint.: currently ok

CROSS-REFERENCE TO R…
REFERENCE TO A SEQUE…
BACKGROUND OF THE IN…
SUMMARY OF THE INVEN…
BRIEF DESCRIPTION OF…
DETAILED DESCRIPTION…
Terms
EXPERIMENTAL DETAILS
Example 1: TNR1B-alk…
Example 2: Fabȃ…
Example 3: Fab-alkyn…
Example 4: N-Termina…
Example 5: GLP1-tria…
Example 6: Biologica…
Example 7: N-Termina…
Example 8: DNA-Triaz…
Example 9: N-Termina…
Example 10: Mertansi…
Example 11: N-Termin…
Example 12: N-Termin…
Example 13: GLP1-Dih…
Example 14: Adalilim…
Example 15: Olanzapi…
DISCUSSION

1. A compound having the structure:

A-b - - - Z

wherein A is a biologically active structure of the compound;

wherein Z is a protein component of the compound, which protein component comprises one or more polypeptides, wherein at least one of the one or more polypeptides comprises consecutive amino acids which (i) are identical to a stretch of consecutive amino acids present in a chain of an f_cdomain of an antibody; (ii) bind to an f_creceptor; and (iii) have at their n-terminus a cysteine or a selenocysteine;

wherein b is (a) an organic acid residue or (b) a stretch of consecutive amino acid residues which is, or is present in any of the following sequences: EPKSCDKTHTCPPCP (SEQ ID NO: 213), ERKCCVECPPCP (SEQ ID NO: 214), ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3 (SEQ ID NO: 215), or ESKYGPPCPSC (SEQ ID NO: 216);

wherein the dashed line between b and Z represents a peptidyl linkage between the n-terminal cysteine or selenocysteine of Z and an amino acid residue or an organic acid residue of b; and

wherein the solid line between A and b represents a nonpeptidyl linkage comprising the structure:

##STR00176##

wherein X_ais a chemical structure containing a cyclooctane fused to a dihydropyridazine; and

wherein r_arepresents an organic structure which connects to A and r_brepresents an organic structure which comprises

##STR00177##

and connects to b, wherein X₁is CH or n, and X₂is CH₂or a carbonyl group.

2. The compound according to claim 1, wherein X_ahas the structure:

##STR00178##

wherein r_cis H, alkyl or aryl;

or a tautomer thereof.

3. The compound according to claim 1, wherein r_ais connected to the cyclooctane and r_bis connected to the dihydropyridazine.

4. The compound according to claim 1, wherein the solid line between A and b represents a nonpeptidyl linkage comprising the structure:

##STR00179##

wherein r_cis H, alkyl or aryl;

or a tautomer thereof.

5. The compound according to claim 1, wherein r_ais a bond or an organic structure comprising or consisting of a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties, wherein each moiety is independently selected from the group consisting of [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, a branched residue, C₁-C₁₀alkyl, C₃-C₁₀cycloalkane, C₂-C₁₀alkene, C₅-C₁₀cycloalkene, amine, sulfur, oxygen, succinimide, maleimide, glycerol, triazole, isoxazolidine, C₂-C₅acyl, C₂-C₅acylamino, C₂-C₅acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, an amino acid, an aryl group, a heteroaryl group, a carbamate, a chemical structure containing a cyclooctane fused to a dihydropyridazine, a chemical structure containing a cyclooctene fused to a triazole, a chemical structure containing a cyclooctene fused to a isoxazolidine, a dibenzocyclooctene, a dibenzoazacyclooctene,

##STR00180## ##STR00181##

wherein X₁is CH or n, X₂is CH₂or a carbonyl group, and r₅is an aryl or alkyl group;

wherein [PEG(y)]z is:

##STR00182##

wherein y=1-100 and z=1-10.

6. The compound according to claim 1, wherein r_a

i) is attached to A via a carbon-nitrogen bond or a carbon-sulfur bond;

ii) is attached to A via a carbon-nitrogen bond;

iii) is attached to A via a carbon-nitrogen bond, wherein the carbon-nitrogen bond is an amide bond;

iv) is attached to A via a biologically labile bond;

v) is attached to A via an amide bond between the C-terminal amino acid of A and an amino group in b;

vi) is attached to A via an amide bond between the C-terminal amino acid of A and an amino group in b, wherein the terminal amino acid is cysteine;

vii) is attached to A via a carbon-sulfur bond;

viii) is attached to A via a carbon-sulfur bond formed between r₂and a free thiol;

ix) is is attached to A via a succinimide-sulfur bond;

x) comprises a branched residue; or

xi) is attached to more than one A via the branched residue.

7. The compound according to claim 1, wherein A

i) comprises the structure of a compound that is a drug approved for treating a subject afflicted with a disease;

ii) comprises the structure of an organic compound having a molecular weight less than 1000 Daltons, a DNA aptamer, an RNA aptamer, an oligonucleotide, or a protein that is biologically active;

iii) comprises a primary or a secondary amine;

iv) is linked to b via the primary or secondary amine;

v) is aripiprazole or oseltamivir;

vi) comprises a secondary amine;

vii) is a respiratory drug, an antiasthmatic agent, an analgesic agent, an antidepressant, an antianginal agent, an antiarrhythmic agent, an antihypertensive agent, an antidiabetic agent, an antihistamine, an anti-infective agent, an antibiotic, an antiinflamatory agent, an antiparkinsonism drug, an antipsychotics, an antipyretic agent, an antiulcer agent, an attention deficit hyperactivity disorder (ADHD) drug, a central nervous system stimulant, a decongestant, or a psychostimulant;

viii) is alprenolol, acebutolol, amidephrine, amineptine, amosulalol, amoxapine, amphetaminil, atenolol, atomoxetine, balofloxacin, bamethan, befunolol, benazepril, benfluorex, benzoctamine, betahistine, betaxolol, bevantolol, bifemelane, bisoprolol, brinzolamide, bufeniode, butethamine, camylofine, carazolol, carticaine, carvedilol, cephaeline, ciprofloxacin, clozapine, clobenzorex, clorprenaline, cyclopentamine, delapril, demexiptiline, denopamine, desipramine, desloratadine, diclofenac, dimetofrine, dioxadrol, dobutamine, dopexamine, doripenem, dorzolamide, droprenilamine, duloxetine, eltopraZine, enalapril, enoxacin, epinephrine, ertapenem, esapraZole, esmolol, etoxadrol, fasudil, fendiline, fenethylline, fenfluramine, fenoldopam, fenoterol, fenproporex, flecamide, fluoxetine, formoterol, frovatriptan, gaboxadol, garenoxacin, gatifloxacin, grepafloxacin, hexoprenaline, imidapril, indalpine, indecainide, indeloxazine hydrochloride, isoxsuprine, ispronicline, labetalol, landiolol, lapatinib, levophacetoperane, lisinopril, lomefloxacin, lotrafiban, maprotiline, mecamylamine, mefloquine, mepindolol, meropenem, metapramine, metaproterenol, methoxyphenamine, dextrorotary methylphenidate, methylphenidate, metipranolol, metoprolol, mitoxantrone, mivazerol, moexipril, moprolol, moxifloxacin, nebivolol, nifenalol, nipradilol, norfloxacin, nortriptyline, nylidrin, olanZapine, oxamniquine, oxprenolol, oxyfedrine, paroxetine, perhexyline, phenmetrazine, phenylephrine, phenylpropylmethylamine, pholedrine, picilorex, pimethylline, pindolol, pipemidic acid, piridocaine, practolol, pradofloxacin, pramipexole, pramiverin, prenalterol, prenylamine, prilocalne, procaterol, pronethalol, propafenone, propranolol, propylhexedrine, protokylol, protriptyline, pseudoephedrine, reboxetine, rasagiline, (r)-rasagiline, repinotan, reproterol, rimiterol, ritodrine, safinamide, salbutamol/albuterol, salmeterol, sarizotan, sertraline, silodosin, sotalol, soterenol, sparfloxacin, spirapril, sulfinalol, synephrine, tamsulosin, tebanicline, tianeptine, tirofiban, tretoquinol, trimetazidine, troxipide, varenicline, vildagliptin, viloxazine, viquidil or xamoterol;

ix) comprises a protein that is biologically active;

x) comprises a secreted protein;

xi) comprises an extracellular domain of a protein

xii) is biologically active such that it has target-binding activity;

xiii) is an independently-folding protein or a portion thereof;

xiv) is a glycosylated protein;

xv) comprises intra-chain disulfide bonds;

xvi) binds a cytokine;

xvii) binds to a cytokine, wherein the cytokine is TNFα;

xviii) comprises Atrial Natriuretic Peptide (ANP), Calcitonin, Corticotropin Releasing Hormone (CRH), Endothelin, Exenatide, Gastric Inhibitory Peptide (GIP), Glucagon-Like Peptide-1 (GLP-1), Glucagon-Like Peptide-2 (GLP-2), an analog of GLP-1 or GLP-2, Glucagon Vasoactive Intestinal Peptide (GVIP), Ghrelin, Peptide YY or Secretin, or a portion thereof;

xix) comprises a stretch of consecutive amino acids in the sequence HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRG (SEQ ID NO: 202);

xx) comprises at least one stretch of consecutive amino acids which are identical to a stretch of consecutive amino acids present in the heavy chain of a Fab or a Fab′ of an antibody;

xxi) comprises at least one at least one stretch of consecutive amino acids which are identical to a stretch of consecutive amino acids present in the light chain of a Fab or a Fab′ of an antibody;

xxii) comprises at least one Fab or Fab′ of an antibody, or a portion of at least one Fab or Fab′;

xxiii) comprises Fab-1 or Fab′1, or a portion thereof of an antibody;

xxiv) comprises Fab-2 or Fab′2, or a portion thereof of an antibody;

xxv) comprises two Fab or Fab′ hands of an antibody;

xxvi) comprises at least one stretch of consecutive amino acids which are identical to a stretch of consecutive amino acids present in a single chain antibody; or

xxvii) comprises at least one stretch of consecutive amino acids which are identical to a stretch of consecutive amino acids present in a TNFα receptor.

8. The compound according to claim 1, wherein Z comprises one C, wherein C is a first polypeptide, which first polypeptide comprises consecutive amino acids which (i) are identical to a stretch of consecutive amino acids present in a chain of an f_cdomain of an antibody; (ii) bind to an f_ereceptor; and (iii) have at their n-terminus a sequence selected from the group consisting of a cysteine, selenocysteine, CP, CPXCP (where X═P, r, or S) (SEQ ID NOs: 206-208), CDKTHTCPPCP (SEQ ID NO: 209), CVECPPCP (SEQ ID NO: 210), CCVECPPCP(SEQ ID NO: 211) and CDTPPPCPRCP (SEQ ID NO: 212), and wherein b is linked to Z via a peptidyl linkage between the n-terminal cysteine or selenocysteine of first polypeptide component C and an amino acid residue or an organic acid residue of b.

9. The compound according to claim 8, wherein C

i) comprises consecutive amino acids which (i) are identical to a stretch of consecutive amino acids present in a chain of an f_cdomain of an antibody; (ii) bind to an f_creceptor; and (iii) have at their n-terminus a sequence comprising a naturally occurring cysteine selected from the group consisting of CP, CPXCP (where X═P, r, or S) (SEQ ID NOs: 206-208), CDKTHTCPPCP (SEQ ID NO: 209), CVECPPCP (SEQ ID NO: 210), CCVECPPCP (SEQ ID NO: 211) and CDTPPPCPRCP (SEQ ID NO: 212);

ii) is a polypeptide component of the compound, which polypeptide component comprises consecutive amino acids which (i) are identical to a stretch of consecutive amino acids present in a chain of an f_cdomain of an antibody;

(ii) bind to an f_creceptor; and (iii) have at their n-terminus a sequence comprising a non-naturally occurring cysteine or selenocysteine;

iii) comprises consecutive amino acids which are identical to a stretch of consecutive amino acids present in the chain of an Fc domain of an antibody selected from the group consisting of IgG, IgM, IgA, IgD, and IgE;

iv) comprises consecutive amino acids which are identical to a stretch of consecutive amino acids present in the chain of an Fc6 domain of an antibody

vi) comprises consecutive amino acids which are identical to a stretch of consecutive amino acids present in a chain of an antibody other than a chain of a Fc domain of the antibody;

vi) consecutive amino acids which are identical to a stretch of consecutive amino acids present in a heavy chain of a Fab or a Fab′ of an antibody; or

vii) comprises consecutive amino acids which are identical to a stretch of consecutive amino acids present in the light chain of a Fab or a Fab′ of an antibody.

10. The compound according to claim 8, wherein Z further comprises a second polypeptide, which second polypeptide comprises consecutive amino acids which are identical to a stretch of consecutive amino acids present in a chain of an antibody other than a chain of a Fc domain of the antibody.

11. The compound according to claim 10, wherein the second polypeptide comprises

i) consecutive amino acids which are identical to a stretch of consecutive amino acids present in a heavy chain of a Fab or a Fab′ of an antibody; or

ii) consecutive amino acids which are identical to a stretch of consecutive amino acids present in the light chain of a Fab or a Fab′ of an antibody.

12. The compound according to claim 1, wherein Z

i) comprises an antibody or a portion thereof;

ii) comprises at least one Fab or Fab′ of an antibody, or a portion of the at least one Fab or Fab′

iii) comprises Fab-1 or Fab′1, or a portion thereof of an antibody;

iv) comprises Fab-2 or Fab′2, or a portion thereof of an antibody;

v) comprises two Fab or Fab′ hands of an antibody;

vi) comprises at least one stretch of consecutive amino acids which are identical to a stretch of consecutive amino acids present in a single chain antibody; or

vii) comprises a second polypeptide, and b is linked to Z via a peptidyl linkage between the n-terminal cysteine or selenocysteine of the second polypeptide of Z and an amino acid residue or an organic acid residue of b.

13. The compound according to claim 8, wherein the C-terminus of C

i) comprises a stretch of consecutive amino acids present in a chain of an f_cdomain of an antibody that has been modified; or

ii) is a cysteine, selenocysteine, homocysteine, or homoselenosysteine, or a derivative of cysteine, selenocysteine, homocysteine, or homoselenosysteine.

14. A homodimer or a heterodimer comprising the compound of claim 1.

15. The homodimer or heterodimer of claim 14, wherein each compound of the homodimer or heterodimer

i) is capable of binding to the other by at least one disulfide bond;

ii) is capable of binding to the other by at least one disulfide bond between the C or the second polypeptide of each compound;

iii) is bound to the other by at least one disulfide bond;

iv) is bound to the other by at least one disulfide bond between the C or the second polypeptide of each compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a § 371 national stage of PCT International Application No. PCT/US2015/020458, filed Mar. 13, 2015, claiming the benefit of U.S. Provisional Application No. 61/953,650, filed Mar. 14, 2014, the content of each of which is hereby incorporated by reference into the application.

REFERENCE TO A SEQUENCE LISTING

This application incorporates-by-reference nucleotide and/or amino acid sequences which are present in the file named “201110_86150-PCT-US_Substitute_Sequence_Listing_DH.txt”, which is 507 kilobytes in size, and which was created Nov. 10, 2020 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is contained in the text file filed Nov. 10, 2020 as part of this application.

Throughout this application, various publications are referenced. The disclosures of all referenced publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

Proteins prefer to form compact globular or fibrous structures, minimizing their exposure to solvent. This tendency is inherent both in the polypeptide backbone with its propensity for hydrogen-bonded secondary structure, and in side chain interactions that promote tertiary folding. Thus, previous efforts to introduce “flexibility” into antibodies using peptides have been largely inadequate. For example, it is common to employ combinations of an amino acid that favors solvent interactions (e.g., serine) with one that breaks up helical structure (e.g., glycine). While this approach is useful in making fusion proteins such as single-chain antibody fragments (scFv), the resulting structures are actually quite compact with no evidence of extendibility (for example, see Robert et al, (2009) Engineered antibody intervention strategies for Alzheimer's disease and related dementias by targeting amyloid and toxic oligomers. Protein Eng. Des. Sel. 22, 199-208). Furthermore, such sequences are likely to create additional problems due to their intrinsic immunogenicity and proteolytic susceptibility.

There is a need for new protein compounds, incorporating nonprotein chains, that are both flexible and extendible, as well as processes for producing such compounds.

SUMMARY OF THE INVENTION

The present invention provides a compound having the structure:
A-B - - - Z

wherein A is a biologically active structure of the compound;

wherein Z is a protein component of the compound, which protein component comprises one or more polypeptides, wherein at least one of the one or more polypeptides comprises consecutive amino acids which (i) are identical to a stretch of consecutive amino acids present in a chain of an F_cdomain of an antibody; (ii) bind to an F_creceptor; and (iii) have at their N-terminus a cysteine or a selenocysteine;

wherein B is a chemical structure linking A and Z;

wherein the dashed line between B and Z represents a peptidyl linkage; and

wherein the solid line between A and B represents a nonpeptidyl linkage.

The present invention also provides a compound having the structure:
L-R_a—B - - - Z

wherein Z is a protein component of the compound, which protein component comprises one or more polypeptides, wherein at least one of the one or more polypeptides comprises consecutive amino acids which (i) are identical to a stretch of consecutive amino acids present in a chain of an F_cdomain of an antibody; (ii) bind to an F_creceptor; and (iii) have at their N-terminus a cysteine or a selenocysteine;

wherein B is a chemical structure linking R_aand C;

wherein the dashed line between B and Z represents a peptidyl linkage;

wherein L is selected from the group consisting of:—N₃, an alkyne, a

##STR00001##
group in which R₅is an alkyl or aryl group, a

##STR00002##
group, a tetrazine and a trans-cyclooctene; and wherein R_ais a bond or an organic structure comprising or consisting of a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties, wherein each moiety is independently selected from the group consisting of [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, a branched residue, C₁-C₁₀alkyl, C₃-C₁₀cycloalkane, C₂-C₁₀alkene, C₅-C₁₀cycloalkene, amine, sulfur, oxygen, succinimide, maleimide, glycerol, triazole, isoxazolidine, C₂-C₅acyl, C₂-C₅acylamino, C₂-C₅acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, an amino acid, an aryl group, a heteroaryl group, a carbamate, a chemical structure containing a cyclooctane fused to a dihydropyridazine, a chemical structure containing a cyclooctene fused to a triazole, a chemical structure containing a cyclooctene fused to a isoxazolidine, a dibenzocyclooctene, a dibenzoazacyclooctene,

##STR00003## ##STR00004##

wherein X₁is CH or N, X₂is CH₂or a carbonyl group, and R₅is an aryl or alkyl group;

- wherein [PEG(y)]z is:

##STR00005##

wherein y=1-100 and z=1-10.

The present invention also provides a process for producing a compound having the structure:
A-B - - - Z

wherein A is a biologically active structure of the compound;

wherein Z is a protein component of the compound, which protein component comprises one or more polypeptides, wherein at least one of the one or more polypeptides comprises consecutive amino acids which (i) are identical to a stretch of consecutive amino acids present in a chain of an F_cdomain of an antibody; (ii) bind to an F_creceptor; and (iii) have at their N-terminus a cysteine or a selenocysteine;

wherein B is a chemical structure linking A and Z;

wherein the dashed line between B and Z represents a peptidyl linkage;

wherein the solid line between A and B represents a nonpeptidyl linkage;

which comprises the following steps:

- a) obtaining an A′ which comprises A or a derivative of A, and a first terminal reactive group;
- b) obtaining a B′ which comprises B or a derivative of B, a second terminal reactive group and a third terminal reactive group, wherein the second terminal reactive group is capable of reacting with the first terminal reactive group to form a non-peptidyl linkage;
- c) obtaining a Z′ which comprises Z or a derivative of Z, and a fourth terminal reactive group, wherein the fourth terminal reactive group is capable of reacting with the third terminal reactive group to form a peptidyl linkage; and
- d) reacting A′, B′ and Z′ in any order to produce the compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the preparation of alkyne-modified TNR1B by cleavage of a TNR1B-intein fusion protein with cystyl-propargylamide. The intein by-product is removed by chitin chromatography. Azide-modified TNR1B and cycloalkyne-modified TNR1B are similarly prepared using cystyl-3-azidopropylamide, and various cyclooctyne (eg. DIBAC) derivatives of cysteine, respectively.

FIG. 2 shows the cleavage of TNR1B by (1) cysteine, (2) cysteine+mercaptoethane sulfonate (MESNA), (3) cystyl-propargylamide, (4) cystyl-propargylamide+MESNA, and (5) MESNA. All compounds were used at 50 mM concentration.

FIG. 3 shows the preparation of azide-modified Fc6 by ligation (peptidyl) of the Fc6 dimer and azide-DKTHT-thioester (Table 1). DKTHT is SEQ ID NO: 220.

FIG. 4 shows the preparation of azide-modified Fc6 by ligation (peptidyl) of the Fc6 dimer and azide-PEG-DKTHT-thioester (Table 1). Cycloalkyne-modified Fc is similarly prepared by using DIBAC-PEG₁₂-thioester. DKTHT is SEQ ID NO: 220.

FIG. 5 shows SDS-PAGE analysis (reducing conditions) of (1) unmodified Fc6, (2) the Az-DKTHT-Fc6 reaction product of FIG. 3, and (3) the Az-PEG₄-DKTHT-Fc6 reaction product of FIG. 4.

FIG. 6 shows the synthesis of TNR1B-alkyne-azide-Fc6 by ligation (non-peptidyl) of alkyne-modified TNR1B and Az-DKTHT-Fc6. DKTHT is SEQ ID NO: 220.

FIG. 7 shows the synthesis of TNR1B-alkyne-azide-PEG_n-Fc6 by ligation (non-peptidyl) of alkyne-modified TNR1B and azide-PEG_n-DKTHT-Fc6. In this example, n=4. DKTHT is SEQ ID NO: 220.

FIG. 8 shows SDS-PAGE analysis (reducing conditions) of (1) alkyne-modified TNR1B alone, (2) alkyne-modified TNR1B+Az-DKTHT-Fc6 in the absence of catalyst, (3) alkyne-modified TNR1B+Az-DKTHT-Fc6+catalyst leading to the product of FIG. 6, and (4) dialyzed alkyne-modified TNR1B+Az-DKTHT-Fc6+catalyst leading to increased formation of the product of FIG. 6 (5) alkyne-modified TNR1B+Az-PEG₄-DKTHT-Fc6 in the absence of catalyst, (6) alkyne-modified TNR1B+Az-PEG₄-DKTHT-Fc6+catalyst leading to the product of FIG. 7, and (7) dialyzed alkyne-modified TNR1B+Az-PEG₄-DKTHT-Fc6+catalyst leading to increased formation of the product of FIG. 7. The arrows correspond to (a) Mr ˜75,000, (b) Mr ˜65,000, (c) Mr ˜43,000, and (d) Mr ˜28,000. DKTHT is SEQ ID NO: 220.

FIG. 9 shows SDS-PAGE analysis (reducing conditions) of (1) TNF1B-Fc fusion protein (etanercept) alone, (2) alkyne-modified TNR1B+Az-DKTHT-Fc6+catalyst leading to the product of FIG. 6, (3) TNF1B-Fc fusion protein (etanercept), and (4) alkyne-modified TNR1B+Az-PEG₄-DKTHT-Fc6 leading to the product of FIG. 7. DKTHT is SEQ ID NO: 220. The arrows correspond to (a) Mr ˜75,000, (b) Mr ˜65,000, (c) Mr ˜43,000, and (d) Mr ˜28,000.

FIG. 10 shows SDS-PAGE analysis (reducing conditions) of (1) unmodified Fc6+catalyst, (2) alkyne-modified TNR1B+unmodified Fc6+catalyst (3) Az-DKTHT-Fc6+catalyst, (4) alkyne-modified TNR1B+Az-DKTHT-Fc6+catalyst leading to the product of FIG. 6, and (5) alkyne-modified TNR1B alone. The arrows correspond to (a) Mr ˜75,000, (b) Mr ˜65,000, (c) Mr ˜43,000, (d) Mr ˜28,000, and (e) Mr ˜27,000. DKTHT is SEQ ID NO: 220.

FIG. 11 shows tryptic peptides identified by LC/MS in the TNR1B-alkyne-azide-DKTHT-Fc6 product (Mr ˜75,000) of FIG. 10. The underlined peptide sequences are those identified by LC/MS that are derived from the parent TNR1B (upper, SEQ ID NO: 204) and Fc6 (lower, SEQ ID NO: 205) sequences. DKTHT is SEQ ID NO: 220.

FIG. 12 shows SPR analysis of TNF-α binding by the TNR1B-alkyne-azide-DKTHT-Fc6 (left panel) and TNR1B-alkyne-azide-PEG₄-DKTHT-Fc6 (right panel) reaction products of FIG. 9. The kinetic binding data are summarized in Table 2. DKTHT is SEQ ID NO: 220.

FIG. 13 shows the preparation of adalimumab Fab′ in a three-step process: 1) IdeS cleavage to the Fab′2+Fc′ fragments, 2) Protein A chromatography to remove the Fc′ fragment, and 3) mild reduction of the Fab′2 fragment to the Fab′ fragment with 2-mercaptoethylamine (MEA).

FIG. 14 shows SDS-PAGE analysis of (1) adalimumab, (2) adalimumab after IdeS cleavage, (3) adalimumab Fab′2 after Protein A purification, (4) adalimumab Fab′ after MEA treatment of the Protein A purified Fab′2, (5) adalimumab Fab′2 after Protein A purification, and (6) adalimumab Fab′ after MEA treatment of the Protein A purified Fab′2. The samples in lanes 1, 2, 5 and 6 were analysis under reducing conditions; while the samples in lanes 3 and 4 were analyzed under non-reducing conditions. The arrows correspond to the (a) heavy chain, (b) heavy chain Fc′ fragment, (c) heavy chain Fd′ (variable region-containing) fragment, and (d) light chain.

FIG. 15 shows the preparation of cycloalkyne-modified Fab′ by the reaction of adalimumab Fab′ with DIBAC-PEG_y-Lys(Mal). In this example, PEGy-PEG₁₂.

FIG. 16 shows SDS-PAGE analysis (non-reducing conditions) of the synthesis and purification of cycloalkyne-modified adalimumab Fab′. Upper panel shows the reaction at (1-6) pH 7.4 and (7-12) pH 7.0. The DIBAC-PEG_y-Lys(Mal) to Fab′ ration was (1) 0, (2) 10:1, (3) 5:1, (4) 2.5:1, (5) 1.2:1, (6) 0.6:1, (7) 0, (8) 10, (9) 5, (10) 2.5, (11) 1.2, and (12) 0.6:1. The lower panel shows (1) unreacted Fab′, (2) through (12) Protein L flow-through fractions containing only the cycloalkyne-modified Fab′.

FIG. 17 shows SDS-PAGE analysis (reducing conditions) of (1) Fc6, (2) Az-DKTHT-Fc6, (3) Az-PEG₁₂-DKTHT-Fc6, (4) Az-PEG₂₄-DKTHT-Fc6, and (5) Az-PEG₃₆-DKTHT-Fc6. DKTHT is SEQ ID NO: 220.

FIG. 18 shows size-exclusion chromatography of (a) Az-PEG₃₆-DKTHT-Fc6, (b) Az-PEG₂₄-DKTHT-Fc6, (c) Az-PEG₁₂-DKTHT-Fc6, (d) Az-DKTHT-Fc6, and (e) Fc6. DKTHT is SEQ ID NO: 220.

FIG. 19 shows the synthesis of Fab′-PEGy-alkyne-azide-PEGx-Fc6 by ligation (non-peptidyl) of cycloalkyne-modified adalimumab Fab′ and azide-modified Fc6.

FIG. 20 shows the Fab′-PEGy-alkyne-azide-PEGx-Fc6 product series.

FIG. 21 shows SDS-PAGE analysis of (1) adalimumab whole antibody, (2) adalimumab Fab′, (3) Fab′-PEG₁₂-alkyne, (4) Fab′-PEG₁₂-alkyne+Az-DKTHT-Fc6, (5) Az-DKTHT-Fc6, (6) Fab′-PEG₁₂-alkyne+Az-PEG₁₂-DKTHT-Fc6, (7) Az-PEG₁₂-DKTHT-Fc6, (8) Fab′-PEG₁₂-alkyne+Az-PEG₂₄-DKTHT-Fc6, (9) Az-PEG₂₄-DKTHT-Fc6 alone, (10) Fab′-PEG₁₂-alkyne+Az-PEG₃₆-DKTHT-Fc6, (11) Az-PEG₃₆-DKTHT-Fc6, and (12) Fc6. Samples were run under reducing conditions (upper panel) and non-reducing conditions (lower panel). In the upper panel the arrow shows (a) Fab′-PEGy-alkyne-azide-PEGx-Fc6 heavy chain. In the lower panels the arrows show (a) two-handed Fab′-PEGy-alkyne-azide-PEGx-Fc6 molecules, and (b) one-handed Fab′-PEGy-alkyne-azide-PEGx-Fc6 molecules. DKTHT is SEQ ID NO: 220.

FIG. 22 shows size-exclusion chromatography (SEC) of two-handed reaction products: (a) Fab′-PEG₁₂-alkyne-azide-PEGa₆-DKTHT-Fc6, (b) Fab′-PEG₁₂-alkyne-azide-PEG₂₄-DKTHT-Fc6, (c) Fab′-PEG₁₂-alkyne-azide-PEG₁₂-DKTHT-Fc6, (d) Fab′-PEG₁₂-alkyne-azide-DKTHT-Fc6, and (e) whole adalimumab. DKTHT is SEQ ID NO: 220.

FIG. 23 shows the inhibition of TNF-α cytotoxity on WEHI cells by reaction products. The upper panel shows the (a) Fc6 control, (b) cycloalkyne-modified Fab′, (c) Fab′-PEG₁₂-alkyne-azide-DKTHT-Fc6, and (d) Fab′-PEG₁₂-alkyne-azide-PEG₁₂-DKTHT-Fc6. The lower panel shows (a) Fc6 control, (b) cycloalkyne-modified Fab′, (c) Fab′-PEG₁₂-alkyne-azide-PEG₂₄-DKTHT-Fc6, and (d) Fab′-PEG₁₂-alkyne-azide-PEGa₆-DKTHT-Fc6. DKTHT is SEQ ID NO: 220.

FIG. 24 shows the purified N3-Px-Fc proteins by SDS-PAGE under reducing (left) and non-reducing conditions (right): Fc6 control (lanes a), N3-P0-Fc (lanes b), N3-P12-Fc (lanes c), N3-P24-Fc (lanes d), N3-P36-Fc (lanes e), and N3-P48-Fc (lanes f).

FIG. 25 shows shows the structure and synthesis of the cyclooctyne-modified GLP-1 analog (GLP1-P7-DBCO).

FIG. 26 shows the reaction between GLP1-P7-DBCO and the N3-Px-Fc proteins.

FIG. 27 shows the structure of GLP1-triazole-Fc hybrid immunoglobulins.

FIG. 28 shows the purified GLP1-triazole-Fc hybrid immunoglobulins by SDS-PAGE under reducing conditions (left) and non-reducing conditions (right): Fc6 control (lanes a), GLP1-P4-DN-P0-Fc (lanes b), GLP1-P4-DN-P12-Fc (lanes c), GLP1-P4-DN-P24-Fc (lanes d), GLP1-P4-DN-P36-Fc (lanes e), and GLP1-P4-DN-P48-Fc (lanes f).

FIG. 29 directly compares the GLP1-triazole-Fc hybrid immunoglobulins and N3-Px-Fc proteins by SDS-PAGE under reducing conditions: Fc6 control (lane a), N3-P0-Fc (lane b), GLP1-P4-DN-P0-Fc (lane c), N3-P12-Fc (lane d), GLP1-P4-DN-P12-Fc (lane e), N3-P24-Fc (lane f), GLP1-P4-DN-P24-Fc (lane g), N3-P36-Fc (lane h), GLP1-P4-DN-P36-Fc (lane i), N3-P48-Fc (lane j), GLP1-P4-DN-P48-Fc (lane k).

FIG. 30 compares the induction of cAMP synthesis in GLP-1 receptor expressing cells by GLP1-triazole-Fc hybrid immunoglobulins and GLP-1.

FIG. 31 shows the reaction between DIBAC-PEG11-DIBAC and the N3-Px-Fc proteins.

FIG. 32 shows the structure of the cyclooctyne-Px-Fc proteins.

FIG. 33 shows the DIBAC-P11-DN—P0-Fc reaction product by SDS-PAGE under reducing conditions: Fc6 control (lane b), unpurified reaction product (lanes c-e), the purified N3-P0-Fc protein (lane f), and the purified DIBAC-P11-DN—P0-Fc protein (lane g).

FIG. 34 shows the reaction between azide-modified DNA and the cyclooctyne-Px-Fc proteins.

FIG. 35 shows the structure of DNA-triazole-Fc hybrid immunoglobulins.

FIG. 36 shows the DNA-triazole-Fc hybrid immunoglobulins reaction products by SDS-PAGE under reducing conditions: the 5AzD-let7d oligonucleotide concentration (mg/ml) was as follows: markers (lane a), 0 (lane b), 2.5 (lane c), 1.25 (lane d), 0.063 (lane e), 0.031 (lane f), 0.016 (lane g), 0.08 (lane h).

FIG. 37 shows the structure and synthesis of the trastuzumab variant, cys1H-IgG1, and the azide-modified trastuzumab heavy chain (N₃-Px-Hc).

FIG. 38 shows the structure and synthesis of the trastuzumab variant, cyslL-IgG1, the azide-modified trastuzumab light chain (N₃-Px-Lc).

FIG. 39 shows the structure and synthesis of cyclooctyne-modified DM-1 (DM1-P4-DBCO).

FIG. 40 shows the reaction between cyclooctyne-modified DM-1 and the N₃-Px-Hc proteins.

FIG. 41 shows the structure of DM1-P4-triazole-Px-Hc hybrid immunoglobulins.

FIG. 42 shows the reaction between cyclooctyne-modified DM-1 and the N₃-Px-Lc proteins.

FIG. 43 shows the structure of DM1-P4-triazole-Px-Lc hybrid immunoglobulins.

FIG. 44 shows the reaction between Tetrazine-DBCO and the N₃-Px-Fc proteins.

FIG. 45 shows the structure of tetrazine-modified Fc proteins (Tet-Px-Fc).

FIG. 47 shows the reaction between TCO-PEG12-DBCO and the N₃-Px-Fc proteins.

FIG. 48 shows the structure of transcyclooctene-modified Fc proteins (Tco-Px-Fc).

FIG. 50 shows the reaction products between NH2-PEG23-N3 and DBCO-TT-P12-P36-Fc protein by SDS-PAGE under reducing conditions: the NH2-PEG23-N3 linker concentration (mg/ml) was as follows: 0.12 (lane a), 0.06 (lane b), 0.03 (lane c), 0.015 (lane d), 0.0075 (lane e), 0.0038 (lane f), 0.002 (lane g), 0.001 (lane h), 0 (lane i).

FIG. 51 shows the structure and synthesis of the transcyclooctene-modified GLP-1 analog (GLP1-P6-Tco).

FIG. 52 shows the reaction between GLP1-P6-Tco peptide and the Tet-Px-Fc proteins.

FIG. 53 shows the structure of the GLP1-dihydropyridizine-Fc hybrid immunoglobulins.

FIG. 54 shows the structure and synthesis of the tetrazine-modified GLP-1 analog (GLP1-P6-Tet).

FIG. 55 shows the reaction between GLP1-P6-Tet peptide and the Tco-Px-Fc proteins.

FIG. 56 shows the structure of the GLP1-P6-TT-Px-Fc hybrid immunoglobulins.

FIG. 57 shows the purified GLP1-dihydropyridizine-Fc hybrid immunoglobulins by SDS-PAGE under reducing conditions (left) and non-reducing conditions (right): Fc6 control (lanes a), GLP1-P6-TT-P0-Fc (lanes b), GLP1-P6-TT-P12-Fc (lanes c), GLP1-P6-TT-P24-Fc (lanes d), GLP1-P6-TT-P36-Fc (lanes e), and GLP1-P6-TT-P48-Fc (lanes f).

FIG. 58 directly compares the N3-Px-Fc (I) proteins, the Tet-Px-Fc (II) proteins, and the GLP1-dihydropyridizine-Fc (III) hybrid immunoglobulins by SDS-PAGE under reducing conditions: Fc6 control (lane a), N3-P0-Fc (lane b), Tet-P0-Fc (lane c), GLP1-P6-TT-P0-Fc (lane d), N3-P12-Fc (lane e), Tet-P12-Fc (lane f), GLP1-P6-TT-P12-Fc (lane g), N3-P24-Fc (lane h), Tet-P24-Fc (lane i), GLP1-P6-TT-P24-Fc (lane j), N3-P36-Fc (lane k), Tet-P36-Fc (lane 1), GLP1-P6-TT-P36-Fc (lane m), N3-P48-Fc (lane n), Tet-P48-Fc (lane o), GLP1-P6-TT-P48-Fc (lane p).

FIG. 59 shows a time course for the reaction of GLP1-P7-DBCO with N3-P36-Fc and a time course for the reaction of GLP1-P6-Tco with Tet-P36-Fc.

FIG. 60 compares the induction of cAMP synthesis in GLP-1 receptor expressing cells by GLP1-dihydropyridizine-Fc hybrid immunoglobulins and GLP-1.

FIG. 61 shows the structure and synthesis of the transcyclooctene-modified adalimumab Fab (Fab-P3-Tco).

FIG. 62 shows the reaction between Fab-P3-Tco protein and the Tet-Px-Fc proteins.

FIG. 63 shows the structure of the Fab-dihydropyridizine-Fc hybrid immunoglobulins.

FIG. 65 shows the structure and synthesis of azide-modified and transcyclooctene-modified olanzapine (Ola-P12-Tco).

FIG. 66 shows the reaction between Ola-P12-Tco and the Tet-Px-Fc proteins.

FIG. 67 shows the structure of olanzapine-dihydropyridizine-Fc hybrid immunoglobulins.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a compound having the structure:
A-B - - - Z

wherein A is a biologically active structure of the compound;

wherein Z is a protein component of the compound, which protein component comprises one or more polypeptides, wherein at least one of the one or more polypeptides comprises consecutive amino acids which (i) are identical to a stretch of consecutive amino acids present in a chain of an F_cdomain of an antibody; (ii) bind to an F_creceptor; and (iii) have at their N-terminus a cysteine or a selenocysteine;

wherein B is a chemical structure linking A and Z;

wherein the dashed line between B and Z represents a peptidyl linkage; and

wherein the solid line between A and B represents a nonpeptidyl linkage.

In some embodiments, the cysteine or selenocysteine naturally occurs in the stretch of consecutive amino acids. In some embodiments, the cysteine or selenocysteine does not naturally occur in the stretch of consecutive amino acids.

In some embodiments, the consecutive amino acids have at their N-terminus a sequence selected from the group consisting of a cysteine, selenocysteine, CP, CPXCP (where X=P, R, or S) (SEQ ID NOs: 206-208), CDKTHTCPPCP (SEQ ID NO: 209), CVECPPCP (SEQ ID NO: 210), CCVECPPCP (SEQ ID NO: 211) and CDTPPPCPRCP (SEQ ID NO: 212).

In some embodiments, the F_cdomain of an antibody is a naturally occurring F_cdomain of an antibody.

In some embodiments, the F_cdomain of an antibody is a variant F_cdomain of an antibody.

In some embodiments, the variant F_cdomain of an antibody is a mutated F_cdomain of an antibody.

In some embodiments, the mutated F_cdomain is a substitution mutant.

In some embodiments, the substitution mutant has an amino acid substitution at the N-terminus, the C-terminus, or at a position of the F_cdomain other than the N-terminus or the C-terminus.

In some embodiments, the substitution mutant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 10-20, or 20-50 amino acid sustitutions in the stretch of consecutive amino acids thereof.

In some embodiments, the substitutions are conservative amino acid substitutions.

In some embodiments, the mutated mutated F_cdomain is an amino acid addition mutant.

In some embodiments, the amino acid addition mutant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 10-20, or 20-50 added amino acids in the stretch of consecutive amino acids thereof.

In some embodiments, the mutated F_cdomain is an amino acid deletion mutant.

In some embodiments, the amino acid deletion mutant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 10-20, or 20-50 deleted amino acids in the stretch of consecutive amino acids thereof.

In some embodiments, the consecutive amino acids are identical to a stretch of at least 0, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, or 190 consecutive amino acids present in the chain of the F_cdomain of the antibody.

In some embodiments, the consecutive amino acids are identical to the stretch of amino acids in the hinge region, the CH2 region or the CH3 region of the Fc domain, or a portion thereof.

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00006##

wherein

##STR00007##
in which R₅is an alkyl or aryl group

wherein R₁is H or is part of an additional structure that is a cyclic structure, wherein the additional cyclic structure comprises R₁or a portion of R₁, and may also comprise R₂or a portion of R₂, and the carbon between R₂and the alkene double bond;

with the proviso that if

##STR00008##
R₃is a H; if

##STR00009##
is a triazole ring that comprises

##STR00010##
and if

##STR00011##
is a N-alkyl or aryl substituted isoxazoline ring that comprises

##STR00012##
and

wherein R₂represents an organic structure which connects to one of A or B and R₄represents an organic structure which connects to the other of A or B.

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00013##

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00014##

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00015##

wherein R₁is part of an additional structure that is a cyclic structure, wherein the additional cyclic structure comprises R₁or a portion of R₁, and may also comprise R₂or a portion of R₂, and the carbon between R₂and the alkene double bond.

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00016##

In some embodiments, R₁and R₂are linked via at least one direct bond so as to form a cyclic structure comprising

i) a portion of R₁,

ii) a portion of R₂,

iii) the carbon between R₂and the alkene double bond, and

iv) the alkene double bond.

In some embodiments, R₁is selected from the group consisting of:

##STR00017##

which is optionally substituted at any position.

In some embodiments, R₁is

##STR00018##
which is optionally substituted at any position.

In some embodiments, R₁is

##STR00019##
which is optionally substituted at any position.

In some embodiments, R₁is

##STR00020##
which is optionally substituted at any position.

In some embodiments, the carbon between R₂and the alkene double bond is:

(i) directly bonded to R₂with a single bond and substituted with two substituents independently selected from the group consisting of hydrogen, halogen, optionally substituted benzyl, optionally substituted alkyl or optionally substituted alkoxy; or

(ii) directly bonded to R₂via a double bond and a single bond.

In some embodiments, the carbon between R₂and the alkene double bond is substituted with two hydrogens and directly bonded to R₂with a single bond.

In some embodiments, the carbon between R₂and the alkene double bond is directly bonded to R₂via a double bond and a single bond.

In some embodiments, the carbon between R₂and the alkene double bond is directly bonded to R₂via a double bond and a single bond so as to form a phenyl ring which is optionally substituted at any position.

In some embodiments, R₂is

##STR00021##
wherein R₂is attached to A or B via J, and

wherein J is a bond or an organic structure comprising or consisting of a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties, wherein each moiety is independently selected from the group consisting of [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, a branched residue, C₁-C₁₀alkyl, C₃-C₁₀cycloalkane, C₂-C₁₀alkene, C₅-C₁₀cycloalkene, amine, sulfur, oxygen, succinimide, maleimide, glycerol, triazole, isoxazolidine, C₂-C₅acyl, C₂-C₅acylamino, C₂-C₅acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, an amino acid, an aryl group, a heteroaryl group, a carbamate, a chemical structure containing a cyclooctane fused to a dihydropyridazine, a chemical structure containing a cyclooctene fused to a triazole, a chemical structure containing a cyclooctene fused to a isoxazolidine, a dibenzocyclooctene, a dibenzoazacyclooctene,

##STR00022## ##STR00023##

wherein X₁is CH or N, X₂is CH₂or a carbonyl group, and R₅is an aryl or alkyl group;

wherein [PEG(y)]z is:

##STR00024##

wherein y=1-100 and z=1-10.

In some embodiments, R₂is

##STR00025##
wherein R₂is attached to A or B via J, and

wherein R₂is attached to R₁via the nitrogen atom of R₂, and

##STR00026## ##STR00027##

wherein X₁is CH or N, X₂is CH₂or a carbonyl group, and R₅is an aryl or alkyl group;

wherein [PEG(y)]z is:

##STR00028##

wherein y=1-100 and z=1-10.

In some embodiments, R₂is

##STR00029##

which is optionally substituted at any position,

wherein R₂is attached to R₁via the nitrogen or carbon atom of R₂,

wherein R₂is attached to A or B via J,

and

##STR00030## ##STR00031##

wherein X₁is CH or N, X₂is CH₂or a carbonyl group, and R₅is an aryl or alkyl group;

wherein [PEG(y)]z is:

##STR00032##

wherein y=1-100 and z=1-10.

In some embodiments, R₂is

##STR00033##

which is optionally substituted at any position.

In some embodiments, R₂is

##STR00034##

which is optionally substituted at any position.

In some embodiments, R₂is

##STR00035##

which is optionally substituted at any position.

In some embodiments, R₂is

##STR00036##

which is optionally substituted at any position.

In some embodiments, R₂is

##STR00037##

which is optionally substituted at any position.

In some embodiments, R₂is

##STR00038##

which is optionally substituted at any position.

In some embodiments, R₁and R₂taken together are:

##STR00039## ##STR00040##

which is optionally substituted at any position,

##STR00041## ##STR00042##

wherein X₁is CH or N, X₂is CH₂or a carbonyl group, and R₅is an aryl or alkyl group;

wherein [PEG(y)]z is:

##STR00043##

wherein y=1-100 and z=1-10.

In some embodiments, R₁and R₂taken together are

##STR00044##

which is optionally substituted at any position.

In some embodiments, R₁and R₂taken together are

##STR00045##

which is optionally substituted at any position.

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00046## ##STR00047##

which is optionally substituted at any position,

##STR00048## ##STR00049##

wherein X₁is CH or N, X₂is CH₂or a carbonyl group, and R₅is an aryl or alkyl group;

wherein [PEG(y)]z is:

##STR00050##

wherein y=1-100 and z=1-10.

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00051##

which is optionally substituted at any position.

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00052##

which is optionally substituted at any position.

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00053## ##STR00054##

which is optionally substituted at any position,

##STR00055## ##STR00056##

wherein X₁is CH or N, X₂is CH₂or a carbonyl group, and R₅is an aryl or alkyl group;

wherein [PEG(y)]z is:

##STR00057##

wherein y=1-100 and z=1-10.

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00058##

which is optionally substituted at any position.

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00059##

which is optionally substituted at any position.

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00060## ##STR00061##

which is optionally substituted at any position,

##STR00062## ##STR00063##

wherein X₁is CH or N, X₂is CH₂or a carbonyl group, and R₅is an aryl or alkyl group;

wherein [PEG(y)]z is:

##STR00064##

wherein y=1-100 and z=1-10.

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00065##

which is optionally substituted at any position.

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00066##

which is optionally substituted at any position.

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00067## ##STR00068##

which is optionally substituted at any position,

##STR00069## ##STR00070##

wherein X₁is CH or N, X₂is CH₂or a carbonyl group, and R₅is an aryl or alkyl group;

wherein [PEG(y)]z is:

##STR00071##

wherein y=1-100 and z=1-10.

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00072##

which is optionally substituted at any position.

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00073##

which is optionally substituted at any position.

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00074##

In some embodiments, R₁is H.

In some embodiments, wherein J is an organic structure comprising a [PEG(y)]z group.

In some embodiments, wherein J is an organic structure comprising a polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), or polysaccharide group.

In some embodiments, J is an organic structure comprising a C₁-C₄alkyl group.

In some embodiments, J is an organic structure comprising a succinimide.

In some embodiments, J is an organic structure comprising amine.

In some embodiments, J is an organic structure comprising a succinyl, malonyl, glutaryl, phthalyl or adipoyl.

In some embodiments, J is an organic structure comprising a malonyl.

In some embodiments, J is an organic structure comprising an amino acid.

In some embodiments, J is an organic structure comprising a cysteine.

In some embodiments, J is an organic structure comprising a lysine.

In some embodiments, J is an organic structure consisting of a chain of 3 moieties selected from the group consisting of [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, a branched residue, C₁-C₁₀alkyl, C₃-C₁₀cycloalkane, C₂-C₁₀alkene, C₅-C₁₀cycloalkene, amine, sulfur, oxygen, succinimide, maleimide, glycerol, triazole, isoxazolidine, C₂-C₅acyl, C₂-C₅acylamino, C₂-C₅acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, an amino acid, an aryl group, a heteroaryl group, a carbamate, a chemical structure containing a cyclooctane fused to a dihydropyridazine, a chemical structure containing a cyclooctene fused to a triazole, a chemical structure containing a cyclooctene fused to a isoxazolidine, a dibenzocyclooctene, a dibenzoazacyclooctene,

##STR00075## ##STR00076##

In some embodiments, J is an organic structure consisting of a chain of four moieties selected from the group consisting of [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, a branched residue, C₁-C₁₀alkyl, C₃-C₁₀cycloalkane, C₂-C₁₀alkene, C₅-C₁₀cycloalkene, amine, sulfur, oxygen, succinimide, maleimide, glycerol, triazole, isoxazolidine, C₂-C₅acyl, C₂-C₅acylamino, C₂-C₅acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, an amino acid, an aryl group, a heteroaryl group, a carbamate, a chemical structure containing a cyclooctane fused to a dihydropyridazine, a chemical structure containing a cyclooctene fused to a triazole, a chemical structure containing a cyclooctene fused to a isoxazolidine, a dibenzocyclooctene, a dibenzoazacyclooctene,

##STR00077## ##STR00078##

In some embodiments, J is an organic structure consisting of a chain of five moieties selected from the group consisting of [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, a branched residue, C₁-C₁₀alkyl, C₃-C₁₀cycloalkane, C₂-C₁₀alkene, C₅-C₁₀cycloalkene, amine, sulfur, oxygen, succinimide, maleimide, glycerol, triazole, isoxazolidine, C₂-C₅acyl, C₂-C₅acylamino, C₂-C₅acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, an amino acid, an aryl group, a heteroaryl group, a carbamate, a chemical structure containing a cyclooctane fused to a dihydropyridazine, a chemical structure containing a cyclooctene fused to a triazole, a chemical structure containing a cyclooctene fused to a isoxazolidine, a dibenzocyclooctene, a dibenzoazacyclooctene,

##STR00079## ##STR00080##

In some embodiments, J comprises a [PEG(y)]z group bonded to a lysine.

In some embodiments, J comprises a C₁-C₄acyl group bonded to a succinimide group.

In some embodiments, J comprises a lysine bonded to a C₁-C₄acyl.

In some embodiments, J comprises a [PEG(y)]z group, which is bonded to a glutaryl.

In some embodiments, J is an organic structure consisting of a chain of five moieties selected from the group consisting of [PEG(y)]z, C₂-C₅acyl, succinyl, malonyl, glutaryl, an amino acid, a chemical structure containing a cyclooctane fused to a dihydropyridazine, a chemical structure containing a cyclooctene fused to a triazole, a chemical structure containing a cyclooctene fused to a isoxazolidine, a dibenzocyclooctene, a dibenzoazacyclooctene,

##STR00081## ##STR00082##

In some embodiments, J is a bond.

In some embodiments, J is a cysteine.

In some embodiments, J has the structure:

##STR00083##

wherein n 1-3, m is 1-4, y is 1-100 and z is 1-10.

In some embodiments, J has a linear structure.

In some embodiments, J has a branched structure.

In some embodiments, R₂is

##STR00084##

wherein n 1-3, m is 1-4, y is 1-100 and z is 1-10.

In some embodiments, R₂is

##STR00085##

wherein n 1-3, m is 1-4, y is 1-100 and z is 1-10.

In some embodiments, R₂is

##STR00086##

wherein n 1-3, m is 1-4, y is 1-100 and z is 1-10.

In some embodiments, R₁and R₂taken together are:

##STR00087##

wherein n 1-3, m is 1-4, y is 1-100 and z is 1-10.

In some embodiments, R₁and R₂taken together are:

##STR00088##

wherein n 1-3, m is 1-4, y is 1-100 and z is 1-10.

In some embodiments, R₁and R₂taken together are:

##STR00089##

wherein n 1-3, m is 1-4, y is 1-100 and z is 1-10.

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00090##

wherein [PEG(y)]z is:

##STR00091##

wherein y=1-100 and z=1-10.

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00092##

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00093##

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00094##

wherein [PEG(y)]z is:

##STR00095##

wherein y=1-100 and z=1-10;

wherein [PEG(x)]w is:

##STR00096##

wherein x=1-100 and w=1-10.

In some embodiments, y is 1-20. In some embodiments, y is 21-40. In some embodiments, y is 41-60. In some embodiments, y is 61-80. In some embodiments, y is 30-50. In some embodiments, y is 12, 24, 36 or 48. In some embodiments, z is 1. In some embodiments, z is 0.

In some embodiments, the terminal carbonyl is of the [PEG(y)]z group is part of an amide bond.

In some embodiments, the terminal amine of the [PEG(y)]z group is part of an amide bond.

In some embodiments, R₄is

##STR00097##

wherein x is 1-100, and w is 0-5.

In some embodiments, x is 1-20. In some embodiments, x is 21-40. In some embodiments, x is 41-60. In some embodiments, x is 61-80. In some embodiments, x is 30-50. In some embodiments, x is 12, 24, 36 or 48.

In some embodiments, w is 1. In some embodiments, w is 0.

In some embodiments, R₄has the structure:

##STR00098##

In some embodiments, R₂is attached A to via J, and R₄is attached to B.

In some embodiments, R₂is attached B to via J, and R₄is attached to A.

In some embodiments, R₄is attached to B via the terminal carbonyl carbon.

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00099##

wherein p=0-5, 0-10, 0-50, or 0-100.

In some embodiments, erein R₂is attached to A via a carbon-nitrogen bond or a carbon-sulfur bond.

In some embodiments, R₂is attached to A via a carbon-nitrogen bond.

In some embodiments, the carbon-nitrogen bond is an amide bond.

In some embodiments, R₂is attached to A via a biologically labile bond.

In some embodiments, R₂is attached to A via an amide bond between the C-terminal amino acid of A and an amino group in B.

In some embodiments, the terminal amino acid is cysteine.

In some embodiments, R₂is attached to A via a carbon-sulfur bond.

In some embodiments, R₂is attached to A via a carbon-sulfur bond formed between R₂and a free thiol.

In some embodiments, R₂is attached to A via a succinimide-sulfur bond.

In some embodiments, J comprises a branched residue.

In some embodiments, J is attached to more than one A via the branched residue.

In some embodiments, B comprises a branched residue.

In some embodiments, B is linked to more than one A, each via a nonpeptidyl linkage with the branched residue.

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00100##

wherein X_ais a chemical structure containing a cyclooctane fused to a dihydropyridazine; and

wherein R_arepresents an organic structure which connects to one of A or B and R_brepresents an organic structure which connects to the other of A or B.

In some embodiments, X_ahas the structure:

##STR00101##

wherein R_cis H, alkyl or aryl;

or a tautomer thereof.

In some embodiments, X_ahas the structure:

##STR00102##

wherein R_cis H, alkyl or aryl;

or a tautomer thereof.

In some embodiments, R_ais connected to the cyclooctane and R_bis connected to the dihydropyridazine.

In some embodiments, the solid line between A and B represents a nonpeptidyl linkage comprising the structure:

##STR00103##

wherein R_cis H, alkyl or aryl;

or a tautomer thereof.

In some embodiments, X_ahas the structure:

##STR00104##

wherein R_cis H, alkyl or aryl;

or a tautomer thereof.

In some embodiments, R_cis methyl.

In some embodiments, R_aand R_bare independently a bond or an organic structure comprising or consisting of a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties, wherein each moiety is independently selected from the group consisting of [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, a branched residue, C₁-C₁₀alkyl, C₃-C₁₀cycloalkane, C₂-C₁₀alkene, C₅-C₁₀cycloalkene, amine, sulfur, oxygen, succinimide, maleimide, glycerol, triazole, isoxazolidine, C₂-C₅acyl, C₂-C₅acylamino, C₂-C₅acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, an amino acid, an aryl group, a heteroaryl group, a carbamate, a chemical structure containing a cyclooctane fused to a dihydropyridazine, a chemical structure containing a cyclooctene fused to a triazole, a chemical structure containing a cyclooctene fused to a isoxazolidine, a dibenzocyclooctene, a dibenzoazacyclooctene,

##STR00105## ##STR00106##

wherein X₁is CH or N, X₂is CH₂or a carbonyl group, and R₅is an aryl or alkyl group;

wherein [PEG(y)]z is:

##STR00107##

wherein y=1-100 and z=1-10.

In some embodiments, R and R_bare independently a bond or an organic structure comprising or consisting of a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties, wherein each moiety is independently selected from the group consisting of [PEG(y)]z, C₂-C₅acyl, succinyl, malonyl, glutaryl, an amino acid, a chemical structure containing a cyclooctane fused to a dihydropyridazine, a chemical structure containing a cyclooctene fused to a triazole, a chemical structure containing a cyclooctene fused to a isoxazolidine, a dibenzocyclooctene, a dibenzoazacyclooctene,

##STR00108## ##STR00109##

wherein X₁is CH or N, X₂is CH₂or a carbonyl group, and R₅is an aryl or alkyl group;

wherein [PEG(y)]z is:

##STR00110##

wherein y=1-100 and z=1-10.

In some embodiments, Ra or Rb is attached to A via a carbon-nitrogen bond or a carbon-sulfur bond.

In some embodiments, Ra or Rb is attached to A via a carbon-nitrogen bond.

In some embodiments, the carbon-nitrogen bond is an amide bond.

In some embodiments, Ra or Rb is attached to A via a biologically labile bond.

In some embodiments, Ra or Rb is is attached to A via an amide bond between the C-terminal amino acid of A and an amino group in B.

In some embodiments, the terminal amino acid is cysteine.

In some embodiments, Ra or Rb is is attached to A via a carbon-sulfur bond.

In some embodiments, Ra or Rb is is attached to A via a carbon-sulfur bond formed between R₂and a free thiol.

In some embodiments, wherein Ra or Rb is is attached to A via a succinimide-sulfur bond.

In some embodiments, Ra or Rb comprises a branched residue.

In some embodiments, Ra or Rb is is attached to more than one A via the branched residue.

In some embodiments, the biological activity of A is increased when it is part of a compound or dimer of the invention compared to the biological activity of A when it is not linked to any other structure.

In some embodiments, A comprises the structure of a compound that is a drug approved for treating a subject afflicted with a disease.

In some embodiments, the subject is a mammalian subject.

In some embodiments, the mammalian subject is a human subject.

In some embodiments, A comprises the structure of an organic compound having a molecular weight less than 1000 Daltons, a DNA aptamer, an RNA aptamer, an oligonucleotide, or a protein that is biologically active.

In some embodiments, the oligonucleotide is an antisense oligonucleotide.

In some embodiments, the oligonucleotide is an RNA interference inducing molecule.

In some embodiments, the oligonucleotide encodes an RNA interference inducing molecule.

In some embodiments, A comprises a primary or a secondary amine.

In some embodiments, A is linked to B via the primary or secondary amine.

In some embodiments, A comprises a primary amine.

In some embodiments, A is aripiprazole or oseltamivir.

In some embodiments, A comprises a secondary amine.

In some embodiments, A is a respiratory drug, an antiasthmatic agent, an analgesic agent, an antidepressant, an antianginal agent, an antiarrhythmic agent, an antihypertensive agent, an antidiabetic agent, an antihistamine, an anti-infective agent, an antibiotic, an antiinflamatory agent, an antiparkinsonism drug, an antipsychotics, an antipyretic agent, an antiulcer agent, an attention deficit hyperactivity disorder (ADHD) drug, a central nervous system stimulant, a decongestant, or a psychostimulant.

In some embodiments, A is alprenolol, acebutolol, amidephrine, amineptine, amosulalol, amoxapine, amphetaminil, atenolol, atomoxetine, balofloxacin, bamethan, befunolol, benazepril, benfluorex, benzoctamine, betahistine, betaxolol, bevantolol, bifemelane, bisoprolol, brinzolamide, bufeniode, butethamine, camylofine, carazolol, carticaine, carvedilol, cephaeline, ciprofloxacin, cloZapine, clobenZorex, clorprenaline, cyclopentamine, delapril, demexiptiline, denopamine, desipramine, desloratadine, diclofenac, dimetofrine, dioxadrol, dobutamine, dopexamine, doripenem, dorzolamide, droprenilamine, duloxetine, eltopraZine, enalapril, enoxacin, epinephrine, ertapenem, esapraZole, esmolol, etoxadrol, fasudil, fendiline, fenethylline, fenfluramine, fenoldopam, fenoterol, fenproporex, flecamide, fluoxetine, formoterol, frovatriptan, gaboxadol, garenoxacin, gatifloxacin, grepafloxacin, hexoprenaline, imidapril, indalpine, indecainide, indeloxazine hydrochloride, isoxsuprine, ispronicline, labetalol, landiolol, lapatinib, levophacetoperane, lisinopril, lomefloxacin, lotrafiban, maprotiline, mecamylamine, mefloquine, mepindolol, meropenem, metapramine, metaproterenol, methoxyphenamine, dextrorotary methylphenidate, methylphenidate, metipranolol, metoprolol, mitoxantrone, mivazerol, moexipril, moprolol, moxifloxacin, nebivolol, nifenalol, nipradilol, norfloxacin, nortriptyline, nylidrin, olanZapine, oxamniquine, oxprenolol, oxyfedrine, paroxetine, perhexyline, phenmetrazine, phenylephrine, phenylpropylmethylamine, pholedrine, picilorex, pimethylline, pindolol, pipemidic acid, piridocaine, practolol, pradofloxacin, pramipexole, pramiverin, prenalterol, prenylamine, prilocalne, procaterol, pronethalol, propafenone, propranolol, propylhexedrine, protokylol, protriptyline, pseudoephedrine, reboxetine, rasagiline, (r)-rasagiline, repinotan, reproterol, rimiterol, ritodrine, safinamide, salbutamol/albuterol, salmeterol, sarizotan, sertraline, silodosin, sotalol, soterenol, sparfloxacin, spirapril, sulfinalol, synephrine, tamsulosin, tebanicline, tianeptine, tirofiban, tretoquinol, trimetazidine, troxipide, varenicline, vildagliptin, viloxazine, viquidil or xamoterol.

In some embodiments, A comprises a protein that is biologically active.

In some embodiments, A comprises a secreted protein.

In some embodiments, A comprises an extracellular domain of a protein.

In some embodiments, A is biologically active such that it has target-binding activity.

In some embodiments, the A is an independently-folding protein or a portion thereof.

In some embodiments, A is a glycosylated protein.

In some embodiments, A comprises intra-chain disulfide bonds.

In some embodiments, A binds a cytokine.

In some embodiments, the cytokine is TNFα.

In some embodiments, A comprises Atrial Natriuretic Peptide (ANP), Calcitonin, Corticotropin Releasing Hormone (CRH), Endothelin, Exenatide, Gastric Inhibitory Peptide (GIP), Glucagon-Like Peptide-1 (GLP-1), Glucagon-Like Peptide-2 (GLP-2), an analog of GLP-1 or GLP-2, Glucagon Vasoactive Intestinal Peptide (GVIP), Ghrelin, Peptide YY or Secretin, or a portion thereof.

In some embodiments, A comprises a stretch of consecutive amino acids in the sequence HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRG (SEQ ID NO:202).

In some embodiments, A comprises at least one stretch of consecutive amino acids which are identical to a stretch of consecutive amino acids present in the heavy chain of a Fab or a Fab′ of an antibody.

In some embodiments, A comprises at least one at least one stretch of consecutive amino acids which are identical to a stretch of consecutive amino acids present in the light chain of a Fab or a Fab′ of an antibody.

In some embodiments, A comprises at least one Fab or Fab′ of an antibody, or a portion of the at least one Fab or Fab′.

In some embodiments, A comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the Fab or Fab′ or portion thereof.

In some embodiments, A comprises Fab-1 or Fab′1, or a portion thereof of the antibody.

In some embodiments, A comprises Fab-2 or Fab′2, or a portion thereof of the antibody.

In some embodiments, A comprises two Fab or Fab′ hands of the antibody.

In some embodiments, the Fab or Fab′ is present in adalimumab

In some embodiments, A comprises at least one stretch of consecutive amino acids which are identical to a stretch of consecutive amino acids present in a single chain antibody.

In some embodiments, A comprises at least one stretch of consecutive amino acids which are identical to a stretch of consecutive amino acids present in a TNFα receptor.

In some embodiments, the TNFα receptor is TNR1B.

In some embodiments, a compound of the invention forms part of a homodimer.

In some embodiments, a compound of the invention forms part of a heterodimer.

The present invention provides homodimers comprising compounds of the invention.

The present invention provides heterodimers comprising compounds of the invention.

In some embodiments, each compound of the dimer is capable of binding to the other by at least one disulfide bond.

In some embodiments, each compound of the dimer is capable of binding to the other by at least one disulfide bond between the Z of each compound.

In some embodiments, each compound of the dimer is bound to the other by at least one disulfide bond.

In some embodiments, each compound of the dimer is bound to the other by at least one disulfide bond between the Z of each compound.

In some embodiments, each compound of the dimer is non-covalently bound to the other.

The present invention provides a compound having the structure:
L-R_a—B - - - Z

wherein Z is a protein component of the compound, which protein component comprises one or more polypeptides, wherein at least one of the one or more polypeptides comprises consecutive amino acids which (i) are identical to a stretch of consecutive amino acids present in a chain of an F_cdomain of an antibody; (ii) bind to an F_creceptor; and (iii) have at their N-terminus a cysteine or a selenocysteine;

wherein B is a chemical structure linking R_aand C;

wherein the dashed line between B and Z represents a peptidyl linkage;

wherein L is selected from the group consisting of: —N₃, an alkyne, a

##STR00111##
group in which R₅is an alkyl or aryl group, a

##STR00112##
group, a tetrazine and a trans-cyclooctene; and

wherein R_ais a bond or an organic structure comprising or consisting of a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties, wherein each moiety is independently selected from the group consisting of [PEG(y)]z, polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide, a branched residue, C₁-C₁₀alkyl, C₃-C₁₀cycloalkane, C₂-C₁₀alkene, C₅-C₁₀cycloalkene, amine, sulfur, oxygen, succinimide, maleimide, glycerol, triazole, isoxazolidine, C₂-C₅acyl, C₂-C₅acylamino, C₂-C₅acyloxy, succinyl, malonyl, glutaryl, phthalyl, adipoyl, an amino acid, an aryl group, a heteroaryl group, a carbamate, a chemical structure containing a cyclooctane fused to a dihydropyridazine, a chemical structure containing a cyclooctene fused to a triazole, a chemical structure containing a cyclooctene fused to a isoxazolidine, a dibenzocyclooctene, a dibenzoazacyclooctene,

##STR00113## ##STR00114##

wherein X₁is CH or N, X₂is CH₂or a carbonyl group, and R₅is an aryl or alkyl group;

- wherein [PEG(y)]z is:

##STR00115##